The concentration of purine metabolites was assessed by high-performance liquid chromatography method in neutralized plasma using UV-VIS detection (Merck-Hitachi/Agilent, Japan/United States) according to the method previously described in details by Smolenski et al. (1990) (link) and used by our team (Zielinski et al., 2019 (link)). The system contains high-pressure gradient pump L-6200, 1050 diode array detector and autosampler AS 2000A with a thermostatic cooler set at 4°C. Separations were achieved by the analytical column BDS Hypersil C18, 150 mm × 4.6 mm × 3 μm (Thermo Finnigan, United States) protected with guard column 20 mm × 4 mm (Phenomenex, United States). Quantitative analysis was conducted using the ChemStation data system (Agilent, United States) operating on a PC. The substances identification was based on comparison retention times with standards of Hx and UA. The measurement was done at wavelength 254 nm for Hx and 280 nm for UA by comparison with external standards. The within-run/between-run %CVs were 3.1/4.1, 3.3/4.4 and 2.7/3.2% for Hx, X and UA, respectively.
Guard column
Manufactured by Phenomenex
Sourced in United States
A guard column is a short, disposable column placed in front of the analytical column in a high-performance liquid chromatography (HPLC) system. Its primary function is to protect the analytical column from contamination, thereby extending the column's lifetime and maintaining the integrity of the chromatographic separation.
Automatically generated - may contain errors
Lab products found in correlation
Luna pfp column, by Phenomenex (3 mentions)
Hplc system, by Shimadzu (3 mentions)
Lc 20at, by Shimadzu (3 mentions)
Agilent 1200 infinity hplc, by Agilent Technologies (3 mentions)
Dual agilent jet stream spray source esi, by Agilent Technologies (3 mentions)
Dgu 14a, by Phenomenex (2 mentions)
Rf 10axl, by Phenomenex (2 mentions)
Rezex roa organic acid h 8 column, by Phenomenex (2 mentions)
Agilent 1200 infinity, by Agilent Technologies (2 mentions)
Gemini c18 column, by Phenomenex (2 mentions)
N2 generator, by Parker Hannifin (2 mentions)
Chemstation data system, by Agilent Technologies (1 mentions)
Hypersil bds c18, by Thermo Fisher Scientific (1 mentions)
Cto 10as vp, by Phenomenex (1 mentions)
Sil 10af, by Phenomenex (1 mentions)
Rid 20a, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Synergi polar rp, by Phenomenex (1 mentions)
Nylon filter, by Avantor (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
38 protocols using guard column
1
Quantitative Analysis of Purine Metabolites
2
Tocopherol Analysis in Pomace Oils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tocopherols (Ts) were determined using the previously validated method of Górnaś et al. [52 (link)]. Crab and dessert apple pomace oils (0.1 g) were diluted in 2-propanol to a volume of 10 mL and filtered through an MS® nylon syringe filter with 0.22 µm pore size (Membrane Solutions, Plano, TX, USA) into 1.5 mL glass vials and immediately analyzed using the HPLC system. The chromatographic separation was carried out using the Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan) consisting of a pump (LC-10ADvp), a degasser (DGU-14A), a low-pressure gradient unit (FCV-10ALvp), a system controller (SCL-10Avp), an auto injector (SIL-10AF), a column oven (CTO-10ASvp), a fluorescence detector (RF-10AXL) and a Luna PFP column (3 µm, 150 × 4.6 mm) with a guard column (4 mm × 3 mm) (Phenomenex, Torrance, CA, USA). The analysis was done using the following conditions: mobile phase methanol:water (93:7; v/v); flow (1.0 mL min−1); and column oven temperature (40 °C). The total separation time was 13.0 min. The identification and quantification of each of tocochromanols homolog was estimated using a fluorescence detector at an excitation wavelength of 295 nm and emission wavelength of 330 nm.
3
Metabolite Profiling by HPLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The supernatant samples were centrifuged at 15,000 rpm for 10 min and filtered by 0.22 μm filters. The standards for the High-Performance Liquid Chromatography (HPLC) measurement of oxoglutarate, fumarate, malate, lactate, acetate, acetoin, glucose and ethanol were prepared with a set of concentration within 0–50 mM. All standards and samples were measured by HPLC using a Shimadzu instrument (LC-20AT, Prominence, Shimadzu) equipped with an Ion exclusion Rezex ROA-Organic Acid H+(8%) column (300 × 7.8 mm column, Phenomenex), along with a guard column (Phenomenex). Aqueous H2SO4 (5 mM) was used as mobile phase at a flow rate of 0.5 ml/min at 55 °C. A wavelength of 210 nm was used for calibration and analysis with the SPD-20 A UV/VIS detector, and a cell temperature of 40 °C was set for refractive index detector (RID 20 A, Shimadzu).
4
Quantitative LC-MS/MS Metabolite Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LC–MS/MS system connected to Agilent 6470 triple-quadrupole mass spectrometer (Agilent, Wilmington, DE, USA) via electrospray ionization (ESI) interface. The mobile phase consisted of methanol: water (65:35, v:v) with 0.1% formic acid. The analytical column of a Synergi Polar-RP (4 µm particle size, 150 × 2 mm, Phenomenex, Torrance, CA, USA) equipped with an guard column (4 × 2 mm, Phenomenex) was used. The sample injection volume, dwell time, and flow rate were 10 µL, 10 min, and 0.3 mL/min, respectively.
5
Separation of Dibenzocyclooctadiene Lignans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Separation of the dibenzocyclooctadiene lignans was conducted on a Luna C18(2) column (ℓ×i.d. = 2.00×150 mm, thickness = 3.0 µm) and a guard column (ℓ×id = 10×3 mm, Phenomenex Inc., Torrance, CA., U.S.A.) using an HPLC system consisting of a Finnigan Surveyor module separation system and a photodiode-array (PDA) detector (Thermo Electron Co., MA., U.S.A.). The next elution process and instrument setting was carried out according to La Torre et al. [24] . We have described the detailed methods in Text S1 .
6
HPLC Analysis of Metabolites from RNA-seq and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of the samples from the RNA sequencing experiment was performed with a JASCO UV/RI HPLC system (JASCO, Easton, MD, USA). Both, a Rezex ROA-Organic Acid H + (8%) column (Phenomenex, Torrance, CA, USA) and a guard column (Phenomenex, Torrance, CA, USA) were used with 5 mM H2SO4 as eluent with a flow rate of 0.8 mL/min and a 5% methanol wash buffer. Five μL of sample was injected and run for 18 min at 80 °C and 46 bar. Peaks were detected with a refractive index (RI) detector. The HPLC was equilibrated for approximately one hour before each run. Peaks were analysed with the ChromNAV program. Samples were prepared before injection onto the column by filtering through 0.2 μm nylon filters (VWR International, Radnor, PY, USA) and followed by a 2 × dilution with Milli-Q water.
Analysis of the samples from the flow cytometry experiments was performed with a Waters HPLC system (Milford, MA, USA) with an Aminex HPX-87H ion exchange column (Bio-Rad, Hercules, CA, USA). The column temperature was 60 °C, 5 mM H2SO4 flowing at 0.6 mL/min was used as mobile phase and a refractive index detector (Waters model 2414) was used for detection. Chromatograms were analysed with the Empower 3 software (Waters, Milford, MA, USA).
Analysis of the samples from the flow cytometry experiments was performed with a Waters HPLC system (Milford, MA, USA) with an Aminex HPX-87H ion exchange column (Bio-Rad, Hercules, CA, USA). The column temperature was 60 °C, 5 mM H2SO4 flowing at 0.6 mL/min was used as mobile phase and a refractive index detector (Waters model 2414) was used for detection. Chromatograms were analysed with the Empower 3 software (Waters, Milford, MA, USA).
7
HPLC Analysis of Ginseng Extract Composition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to pharmacological evaluation, the AG extract was analyzed using HPLC [20,21] . The HPLC system was a Waters Alliance 2960 instrument (Milford, MA, USA) with a quaternary pump, an automatic injector, a photodiode array detector (Model 996), and Waters Millennium 32 software for peak identification and integration. The separation was carried out on a Prodigy ODS(2) column (250 mm × 3.2 mm inner diameter) with a guard column (3.0 mm × 4.0 mm inner diameter) (Phenomenex, Torrance, CA, USA). For HPLC analysis, a 20-μL sample was injected into the column and eluted at room temperature with a constant flow rate of 1.0 mL/min. For the mobile phase, acetonitrile (solvent A) and water (solvent B) were used. Gradient elution started with 17.5% solvent A and 82.5% solvent B. Elution solvents were then changed to 21% A for 20 min, then to 26% A for 3 min and held for 19 min, at 36% A for 13 min, at 50% A for 9 min, at 95% A for 2 min, and held for 3 min. Lastly, eluting solvents were changed to 17.5% A for 3 min and held for 8 min. The detection wavelength was set at 202 nm. All sample solutions were filtered through a membrane filter (0.2 μm pore size). The content of the constituents were calculated using the standard curves of 13 ginsenosides. The measurement for the content analysis of the AG was performed in triplicate.
8
HPLC-Based Compound Separation Procedure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chromatographic system composed of a Dionex UltiMate 3000 high performance liquid chromatography (HPLC) System (Thermo Fisher Scientific, Waltham, MA, USA), which contained a HPG-3400SD solvent delivery pump, WPS-3000(T)SL Analytical Autosampler, and Dionex UltiMate 3000 RS fluorescence detector. The manipulation of data was carried out by Thermo Fisher Scientific Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS) integration software.
The segregation was achieved using a Luna RP-C18 column (250×4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA) conserved with a guard column (Phenomenex) equipped just before the inlet junction of the analytical column.
The segregation was achieved using a Luna RP-C18 column (250×4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA) conserved with a guard column (Phenomenex) equipped just before the inlet junction of the analytical column.
9
HPLC Analysis of MK-2048 in PNP Films
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dionex Ultimate 3000 HPLC system (Thermo Fisher, Waltham, MA, USA) and Chromeleon data acquisition system was used. The column used was the Xbridge C18 HPLC column (5 µm, 2.1 × 50 mm, Waters, Milford, MA, USA) with Guard Column (Phenomenex, Torrance, CA, USA). The mobile phases consist of (A) aqueous phase: 0.1% of formic acid in Milli-Q water, and (B) organic phase: acetonitrile. Gradient elution with a 1 mL/min flow rate was used for separation. The gradient is as follows: at 0–2 min, 10% B; 2–6 min, a linear change to 50% B; 6–7 min, 50% B; 7–8 min, a linear change to 10% B; 8–11 min, 10% B. 10 µL of the sample was injected in duplicate. MK-2048 can be detected at a wavelength of 334 nm. Drug Loading (DL%) was calculated using the ratio of MK-2048 detected in PNPs/PNP films to the measured PNP/ PNP film weights, respectively.
10
Reversed-Phase HPLC Separation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chromatographic separation was performed on an Agilent 1200 Infinity HPLC chromatograph (Agilent Technologies, Santa Clara, CA, USA) using a thermostated (20 °C) Gemini® chromatographic column (3 µm i.d. C18 with TMS endcapping, 110 Å, 150 x 2 mm) and guard column (Phenomenex Inc, Torrance, CA, USA). The flow rate was set at 0.3 mL/min for the mobile phase A (water containing 0.1% formic acid (v/v)) and mobile phase B (0.1% formic acid in acetonitrile(v/v)). Both mobile phases were mixed in the following gradient program: 5 min, 0% B; 20 min, 66% B; 35 min, 95% B. The stop time was set at 35 min.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!